Methods for Improving Human Gut Microbiome Data by Reducing Variability through Sample Processing and Storage of Stool

PLoS One. 2015 Aug 7;10(8):e0134802. doi: 10.1371/journal.pone.0134802. eCollection 2015.


Gut microbiome community analysis is used to understand many diseases like inflammatory bowel disease, obesity, and diabetes. Sampling methods are an important consideration for human microbiome research, yet are not emphasized in many studies. In this study, we demonstrate that the preparation, handling, and storage of human faeces are critical processes that alter the outcomes of downstream DNA-based bacterial community analyses via qPCR. We found that stool subsampling resulted in large variability of gut microbiome data due to different microenvironments harbouring various taxa within an individual stool. However, we reduced intra-sample variability by homogenizing the entire stool sample in liquid nitrogen and subsampling from the resulting crushed powder prior to DNA extraction. We experimentally determined that the bacterial taxa varied with room temperature storage beyond 15 minutes and beyond three days storage in a domestic frost-free freezer. While freeze thawing only had an effect on bacterial taxa abundance beyond four cycles, the use of samples stored in RNAlater should be avoided as overall DNA yields were reduced as well as the detection of bacterial taxa. Overall we provide solutions for processing and storing human stool samples that reduce variability of microbiome data. We recommend that stool is frozen within 15 minutes of being defecated, stored in a domestic frost-free freezer for less than three days, and homogenized prior to DNA extraction. Adoption of these simple protocols will have a significant and positive impact on future human microbiome research.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteria / classification
  • DNA / metabolism
  • Feces / microbiology*
  • Freezing
  • Gastrointestinal Microbiome*
  • Humans
  • Preservation, Biological*
  • RNA / metabolism
  • Real-Time Polymerase Chain Reaction
  • Specimen Handling / methods*
  • Temperature


  • RNA
  • DNA

Grant support

N.T. was an I.K. Barber Undergraduate Research Awardee; S.K.G. was a Canadian Association of Gastroenterology Awardee and M.J. was a National Science and Engineering Research Council Awardee. This work was supported by grants funded through Natural Science and Engineering Research Council and Crohn’s and Colitis Canada to D.L.G.